1. Field of the Invention
The present disclosure relates to tissue systems, methods of producing tissue systems and methods of treatment using tissue systems comprising conjunctival cells for restoring damaged or diseased ocular surfaces. The tissue system of the present disclosure comprises mammalian conjunctival cells, preferably progenitor conjunctival cells derived from the fornix region of the conjunctiva. The present disclosure in particular relates to the culturing of progenitor conjunctival cells on a suitable support material such as amniotic membrane under controlled culture conditions.
2. Description of Related Art
Stem cells are responsible for cellular replacement and tissue regeneration throughout the life of an organism. Stem cells are unspecialized cells that give rise to specialized cells, and have extensive proliferation potential. Depending on the type of stem cell, these cells may differentiate into several cell lineages and/or repopulate a tissue upon transplantation. Embryonic stem (ES) cells are quintessential stem cells with unlimited self-renewal and pluripotent potential, and are derived from the inner cell mass of a blastocyst-stage embryo. Adult stem cells are specialized undifferentiated stem cells, which, after birth and throughout adulthood, retain the ability to replace cells and regenerate tissues in an organism. It is generally understood that adult stem cells, as compared to ES cells, have less self-renewal ability, and although they may differentiate into multiple lineages, are not generally described as pluripotent. Cell therapy has the potential to treat any disease that is associated with cell dysfunction or damage, including but not limited to the potential for manipulating stem cells, whether ES cells or adult stem cells, to repair or replace diseased or damaged tissue. This potential has generated a great deal of excitement in the scientific, medical, and biotechnology communities.
Adult stem cells (also referred to as “tissue-specific stem cells”) have been found in various tissues of the adult body, including bone marrow (Weissman, Science 287:1442-1446, 2000), neural tissue (Gage, Science 287:1433-1438, 2000), gastrointestinal tissue (Potten, Phil Trans R Soc Lond. B. 353:821-830, 1998), epidermal tissue (Watt, Phil Trans R Soc Lond B. 353:831, 1997), hepatic tissue (Alison and Sarraf, J Hepatol. 29:678-683, 1998), and mesenchymal tissue (Pittenger et al., Science 284:143-147, 1999). Adult stem cells that are found in the comeoscleral limbus of the mammalian eye are essential for the maintenance of a healthy ocular surface, and participate in the dynamic equilibrium of healthy ocular and corneal surfaces.
The surface of the eye consists of the cornea, the conjunctiva, and the border between the two, which is known as the comeoscleral junction, or limbus. The optical surface has two basic epithelial surfaces, corneal epithelium and conjunctival epithelium. The conjunctiva is a two to three layer epithelium that extends from the mucocutaneous junction, beginning at the eyelashes on to the inner surface of the eyelids, over the ocular surface on the eye proper and ending at the limbus. Stem cells that continually replace conjunctival epithelium have been shown to reside at the conjunctival fornix. Wei et al., Invest Ophthalmol Vis Sci. 34:1814-28, 1993. These stem cells, which lie at the uppermost and lowermost regions of the conjunctiva at the forniceal junction between the sclera and the eyelids, are known as conjunctival stem cells or progenitor conjunctival cells.
A major role of the conjunctiva is to provide ocular surface hydration and lubrication through the production of tears by mucin-producing goblet cells, which are highly specialized epithelial cells that are interspersed between the conjunctival epithelial cells. Mucins are highly glycosylated proteins secreted primarily by goblet cells present in the conjunctiva. The conjunctival surface integrity is influenced by the level of ocular mucin that is secreted by goblet cells. Mucin production is the main characteristic feature of conjunctival cells. The multiple layers of stratified conjunctival epithelium along with the interspersed goblet cells together are continuously regenerated maintaining the functional integrity of the ocular surface (Kessing, S V, Acta Ophthalmol 44:439-453, 1966; Lemp, et al., Arch Ophthalmol. 83:89-94, 1970).
Mucin deficiency is detected in alkali burns, chemical and thermal burns, Stevens-Johnson Syndrome (SJS), neurotrophic keratitis and Ocular Cicatricial Pemphigoid (OCP). See Gilbard and Rossi, Ophthalmol. 97:308-312; 1990; Lemp, Int Ophthalmol Clin. 13:185-189, 1973; Tseng et al., Ophthalmol. 91:545-552, 1984. The conjunctiva also provides a smooth and wet cellular surface to support the tear film on the corneal surface, which in turn results in an optically clear optical surface and clear vision. Thus, the conjunctiva supports the health of the corneal epithelium. Because the corneal epithelium is dependent on a healthy conjunctival surface to maintain clear vision, many ocular surface diseases initiate with conjunctival damage, followed by secondary limbal and corneal damage. Stratifying epithelia are self-renewing tissues that undergo constant reorganization, necessitated by a continuous loss of terminally differentiated superficial cells, balanced by basal cell proliferation. Therefore, in order to maintain a healthy epithelial structure, a critical balance between cell proliferation and differentiation must be achieved at all times.
Tissue culture techniques have been widely employed for stratified epithelia such as epidermis and cornea to begin to identify those factors that may be important during keratinocyte proliferation and maturation. Researchers in this area have utilized rabbit or bovine conjunctival epithelial cells in culture, and several models for the culture of normal human conjunctival keratinocytes have been developed. Primary human conjunctival keratinocytes have been propagated in long-term culture from cadaver eyes using serum containing culture medium and, in some cases, a fibroblast feeder layer. Lindberg et al., Invest Ophthalmol Vis Sci. 34:2672-2679, 1993; Tsai et al., Invest Ophthalmol Vis Sci. 35:2865-2875, 1994.
In the clinical context of ocular injuries, conjunctival involvement has been estimated by dividing the bulbar and forniceal conjunctiva into quadrants and determining the area involved. The involvement of the bulbar and forniceal conjunctiva is considered significant for the eventual outcome after ocular injury or disease. Argüeso et al., Invest Ophthalmol Vis Sci. 43:1004-1011, 2002; Dua et al., Br J Ophthalmol. 85:1379-1383, 2001; Pfister, Opthalmology 90:1246-53, 1983. For example, in grade 4, 5 and 6 ocular burns, in which the surviving corneal and conjunctival epithelium is thin to absolutely zero, the desirable ocular management may be to attempt a restorative and reconstructive intervention by re-establishing both corneal and conjunctival epithelial cover, one after the other (see Dua, Br J Opthalmol. 82: 1407-11, 1998). In Nakamura et al., Invest Ophthalmol Vis Sci 44(1):106-116, 2003, re-establishment of the corneal mileu was attempted by transplanting cultured limbal stem cells on human amniotic membrane (HAM) with good prognosis. See also Shimazaki et al., Ophthalmology 109(7):1285-1290, 2003; Tseng et al., Arch Ophthalmol. 116:431-41, 1998; Koizumi et al., Ophthalmology 108(9):1569-1574, 2001. There are also reports in the literature on conjunctival autografts and culturing progenitors from bulbar and forniceal regions on HAM (see Grueterich et al., Surv Ophthalmol. 48(6):631-46, 2003; Wei et al., Invest Ophthalmol Vis Sci. 34:1814-1828, 1993).
With respect to conjunctival tissue equivalents, U.S. Pat. No. 7,049,139 discloses culturing conjunctival cells on HAM in a multi-step process using three different media to produce a conjunctival tissue equivalent. The three different steps used to prepare the tissue equivalent include culturing the conjunctival cells in a primary culture media, a proliferative culture media, and a differentiative culture media. Two of the three media types disclosed include cholera toxin as one of the ingredients of the medium. Cholera toxin, which is a potential carcinogen, can cause eye irritation. The multi-step process was used to enhance the stability of the tissue equivalent for clinical transplantation. The efficiency of a conjunctival tissue equivalent cultivated in serum-free conditions on HAM was studied after the graft was transplanted in seven patients with different conjunctival surface disorders. Epithelisation and graft integrity was studied. Tan et al., Transplantation 77(11):1729-1734, 2004. See also, Tan et al., U.S. Pat. No. 7,049,139; Ang et al., (2004) Invest Ophthalmol Vis Sci. 45 (6):1789-1795. U.S. Pat. No. 7,049,139 requires three steps to produce a conjunctival tissue equivalent, using a culturing media, a proliferation media, and a differentiation media. The media employed for differentiation includes a high calcium concentration, as well as choleratoxin.
Most studies of tissue equivalents or tissue systems have focused on the ability to reconstitute a tissue equivalent that bears the structural and functional characteristics of the tissue of origin. This has been achieved by differentiating cells in culture, for example, by modifying the culture conditions and air-lifting. Terminally differentiated cells have limited long-term proliferative capacity, however, which results in a lower regenerative potential after transplantation. In addition, the use of epithelial tissue constructs for ocular surface transplantation requires that cells be sufficiently attached to the underlying substrate so that they are not sloughed off by direct mechanical or shearing forces, during or after surgical transplantation. Therefore, a delicate balance is necessary to preserve the proliferative potential of transplanted cells, while at the same time ensuring that transplanted cells have the necessary functional characteristics of the tissue organ. The ideal tissue construct is one in which transplanted cells possess a long-term regenerative capability for cellular renewal and replacement of tissue.
While tissue equivalents developed may be transplanted by conventional methods, for example suturing the graft onto the eye, this can cause discomfort to the recipient. Further, the act of suturing requires more precision then a suture-less method, since the site of the diseased or damaged eye cannot be clearly visualized. In addition, such transplantations usually leave a scar at the site. Thus, a suture-less transplantation would be preferred over conventional methods necessitates the development of other techniques or cell-based membrane delivery systems that can adhere to the site of transplantation without suture. Pfister and Sommers, Cornea 24(5):593-598, 2005, found that fibrin sealant alone can attach corneal stem cell transplants to the limbal niche.
In response to the need for a more desirable conjunctival tissue equivalent, the present disclosure describes a tissue system with a long-term regenerative capability for cellular renewal and replacement of damaged or diseased tissue on the ocular surface. A particular advantage of the presently disclosed conjunctival tissue system is that it involves a relatively simple culture scheme, in that it employs a single media used for culturing, expansion and differentiation of the conjunctival tissue system, and can avoid the use of toxins such as choleratoxin and high calcium concentration. This simple culturing scheme may minimize contamination problems in cGMP facilities. In addition, the conjunctival tissue system disclosed herein may be introduced onto the ocular surface using a suture-less method.